Il 4 pecy7

For assessment of intracellular cytokine expression in CD4⁺ T cells, the lymphocytes were incubated with 10 μg/mL brefeldin A (Sigma-Aldrich, St. Louis, MO, USA). After 6 hours, cells were harvested and stained with anti-CD4-FITC (eBioscience, Inc., San Diego, CA, USA) for 30 min at room temperature, followed by fixation and permeabilization using the Cytofix/Cytoperm Kit (BD Biosciences, San Diego, CA, USA) according to the manufacturer’s instructions. The intracellular cytokine staining was performed using the following monoclonal antibodies: IFN-γ-PE, IL-4-PEcy7, IL-17-PE, and Foxp3-PEcy7 (eBioscience, Inc., San Diego, CA, USA). Stained cells were analyzed by a cube-8 flow cytometer (Sysmex/Partec, Münster, Germany). Gates were applied to define the populations of interest, and the analysis was carried out using FCS Express 4 (De Novo Software, CA, USA). Isotype controls were used as negative controls.

Lung single-cell suspensions [54 (link)] were stained with a fixable viability marker, stained with an antibody panel (below), and assayed on a FACS Canto II (BD; Franklin Lakes, NJ). For CD4 T cell population identification and determination of IL-33 cell sources, cells were stimulated for 5 hr with phorbol-12-myristate-13-acetate (PMA; 5 ng ml^-1) and ionomycin (500 ng ml^-1) in the presence of a protein transport inhibitor (GolgiPlug; BD) prior to intracellular staining with CD3—eFluor450, CD4—PerCP, IFNγ—PE, and IL-4—PE-Cy7 or CD45—PerCP-Cy5.5, EpCam—APC, CD31—PE-Cy7 (all eBioscience), and IL-33—PE (R&D Systems; Minneapolis, MN). For ILC2 staining, cells were labeled with antibodies to CD3—PacBlue, CD19—PacBlue, CD11c—PacBlue, CD11b—PacBlue, CD49b—PacBlue, F4/80—PacBlue, FcεRI—PacBlue, and Sca-1—PE-Cy7, (all BioLegend; San Diego, CA), ST2—FITC (MD Bioproducts; St. Paul, MN), Thy1.2 (CD90)—biotin (Southern Biotech; Birmingham, AL), Gata3—PE-Cy7 (BD), CD45—PerCP-Cy5.5, CD25—APC-Cy7/PE, and ICOS—APC (all eBioscience). Data were analyzed using FlowJo v10 (S1 Fig).

Human basophils were gated on FcεRIα-FITC/CD123-PerCP/Cy5.5/CD203c-PE (BioLegend, San Diego, CA, USA) positive cells after extracellular and intracellular staining. The expression levels of CD203c-PE, CD62L-APC, FcεRIα-FITC, CCR7-APC, CD63-APC, IL-13-APC, B cell-activating factor (BAFF)-APC (BioLegend, San Diego, CA, USA), IL-4-PE-Cy7, and IL-6-APC (eBioscience, San Diego, CA, USA) in basophils were quantified and expressed as relative fluorescence units (the ratio of mean fluorescence intensity normalized to controls) or as a positive percentage of total basophils.
Mouse basophils were gated on CD49b-APC/IgE-PE (BioLegend, San Diego, CA, USA). The expression levels of the activation marker CD200R-FITC (BioLegend, San Diego, CA, USA) (26 (link)), IL-4-FITC, and IL-6-FITC (eBioscience, San Diego, CA, USA) were quantified and expressed in the same way as for human basophils. A FACScanto™ Π flow cytometer (Becton Dickinson, San Jose, CA, USA) and Lysys II software (Becton Dickinson, San Jose, CA, USA) or FlowJo Software (Tree Star, San Carlos, CA, USA) were used to acquire and analyze the data.